The Role of Fibrogenesis and Extracellular Matrix Proteins in the Pathogenesis of Graves' Ophthalmopathy.

Graves' ophthalmopathy (GO), or thyroid eye disease (TED), is the most frequent extrathyroidal manifestation of Graves' disease (GD). Inflammation and subsequent aberrant tissue remodeling with fibrosis are important pathogenesis. There are many proposed mechanisms and molecular pathways contributing to tissue remodeling and fibrosis in GO, including adipogenesis, fibroblast proliferation and myofibroblasts differentiation, oxidative stress, endoplasmic reticulum (ER) stress, hyaluronan (HA) and glycosaminoglycans (GAGs) accumulation in the extracellular matrix (ECM) and new concepts of epigenetics modification, such as histone modification, DNA methylation, non-coding RNAs, and gut microbiome. This review summarizes the current understanding of ECM proteins and associated tissue remodeling in the pathogenesis and potential mediators for the treatment of GO.

JNK and p38 Inhibitors Prevent Transforming Growth Factor-ß1-Induced Myofibroblast Transdifferentiation in Human Graves' Orbital Fibroblasts.

Transforming growth factor-ß1 (TGF-ß1)-induced myofibroblast transdifferentiation from orbital fibroblasts is known to dominate tissue remodeling and fibrosis in Graves' ophthalmopathy (GO). However, the signaling pathways through which TGF-ß1 activates Graves' orbital fibroblasts remain unclear. This study investigated the role of the mitogen-activated protein kinase (MAPK) pathway in TGF-ß1-induced myofibroblast transdifferentiation in human Graves' orbital fibroblasts. The MAPK pathway was assessed by measuring the phosphorylation of p38, c-Jun N-terminal kinase (JNK), and extracellular-signal-regulated kinase (ERK) by Western blots. The expression of connective tissue growth factor (CTGF), α-smooth muscle actin (α-SMA), and fibronectin representing fibrogenesis was estimated. The activities of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) responsible for extracellular matrix (ECM) metabolism were analyzed. Specific pharmacologic kinase inhibitors were used to confirm the involvement of the MAPK pathway. After treatment with TGF-ß1, the phosphorylation levels of p38 and JNK, but not ERK, were increased. CTGF, α-SMA, and fibronectin, as well as TIMP-1 and TIMP-3, were upregulated, whereas the activities of MMP-2/-9 were inhibited. The effects of TGF-ß1 on the expression of these factors were eliminated by p38 and JNK inhibitors. The results suggested that TGF-ß1 could induce myofibroblast transdifferentiation in human Graves' orbital fibroblasts through the p38 and JNK pathways.

Connective tissue growth factor decreases mitochondrial metabolism through ubiquitin-mediated degradation of mitochondrial transcription factor A in oral squamous cell carcinoma.

BACKGROUND/PURPOSE: Deregulation of metabolic pathways is one of the hallmarks of cancer progression. Connective tissue growth factor (CTGF/CCN2) acts as a tumor suppressor in oral squamous cell carcinoma (OSCC). However, the role of CTGF in modulating cancer metabolism is still unclear. METHODS: OSCC cells stably overexpressing CTGF (SAS/CTGF) and shRNA against CTGF (TW2.6/shCTGF) were established. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were examined by the Seahorse XF24 analyzer. The expression of CTGF and mitochondrial biogenesis related genes was measured by real-time polymerase chain reaction or Western blot analysis. RESULTS: CTGF decreased OCR, ECAR, adenosine triphosphate (ATP) generation, mitochondrial DNA (mtDNA), and mitochondrial transcription factor A (mtTFA) protein expression in OSCC cells. Overexpression of mtTFA restored CTGF-decreased OCR, ECAR, mtDNA copy number, migration and invasion of SAS/CTGF cells. Immunoprecipitation assay showed a higher level of ubiquitinated mtTFA protein after CTGF treatment. MG132, an inhibitor of proteasomal degradation, reversed the effect of CTGF on mtTFA protein expression in SAS cells. CONCLUSION: CTGF can decrease glycolysis, mitochondrial oxidative phosphorylation, ATP generation, and mtDNA copy number by increasing mtTFA protein degradation through ubiquitin proteasome pathway and in turn reduces migration and invasion of OSCC cells. Therefore, CTGF may be developed as a potential additive therapeutic drug for oral cancer in the near future.

Role of AMPK-mediated adaptive responses in human cells with mitochondrial dysfunction to oxidative stress.

BACKGROUND: Mitochondrial DNA (mtDNA) mutations are an important cause of mitochondrial diseases, for which there is no effective treatment due to complex pathophysiology. It has been suggested that mitochondrial dysfunction-elicited reactive oxygen species (ROS) plays a vital role in the pathogenesis of mitochondrial diseases, and the expression levels of several clusters of genes are altered in response to the elevated oxidative stress. Recently, we reported that glycolysis in affected cells with mitochondrial dysfunction is upregulated by AMP-activated protein kinase (AMPK), and such an adaptive response of metabolic reprogramming plays an important role in the pathophysiology of mitochondrial diseases. SCOPE OF REVIEW: We summarize recent findings regarding the role of AMPK-mediated signaling pathways that are involved in: (1) metabolic reprogramming, (2) alteration of cellular redox status and antioxidant enzyme expression, (3) mitochondrial biogenesis, and (4) autophagy, a master regulator of mitochondrial quality control in skin fibroblasts from patients with mitochondrial diseases. MAJOR CONCLUSION: Induction of adaptive responses via AMPK-PFK2, AMPK-FOXO3a, AMPK-PGC-1α, and AMPK-mTOR signaling pathways, respectively is modulated for the survival of human cells under oxidative stress induced by mitochondrial dysfunction. We suggest that AMPK may be a potential target for the development of therapeutic agents for the treatment of mitochondrial diseases. GENERAL SIGNIFICANCE: Elucidation of the adaptive mechanism involved in AMPK activation cascades would lead us to gain a deeper insight into the crosstalk between mitochondria and the nucleus in affected tissue cells from patients with mitochondrial diseases. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research.

Effects of carbonic anhydrase-related protein VIII on human cells harbouring an A8344G mitochondrial DNA mutation.

MERRF (myoclonus epilepsy associated with ragged-red fibres) is a maternally inherited mitochondrial encephalomyopathy with various syndromes involving both muscular and nervous systems. The most common mutation in MERRF syndrome, the A8344G mutation in mtDNA, has been associated with severe defects in the respiratory function of mitochondria. In the present study, we show that there is a significant decrease in CA8 (carbonic anhydrase-related protein VIII) in cybrids harbouring the MERRF A8344G mutation. CA8 deficiency and mutations were found to be associated with a distinctive lifelong gait disorder in wdl (Waddles) mice and novel syndromes characterized by cerebellar ataxia and mental retardation in humans. The results of the present study showed that overexpression of CA8 in MERRF cybrids significantly decreased cell death induced by STS (staurosporine) treatment, suggesting a protective function of CA8 in cells harbouring the A8344G mutation of mtDNA. Interestingly, an increase in the formation of LC3-II (microtubule-associated protein 1 light chain 3-II) was found in the cybrids with down-regulated CA8 expression, suggesting that reduced expression of CA8 leads to autophagy activation. Furthermore, cybrids exhibiting down-regulated CA8 showed increased cytosolic Ca2+ signals and reduced levels of phospho-Akt compared with those in the cybrids with overexpressed CA8, indicating that phospho-Akt is involved in the protection of cells by CA8. Our findings suggest that CA8 is involved in the autophagic pathway and may have a protective role in cultured cells from patients with MERRF. Targeting CA8 and the downstream autophagic pathway might help develop therapeutic agents for treatment of MERRF syndrome in the future.

A Review of Pathophysiology and Therapeutic Strategies Targeting TGF-ß in Graves' Ophthalmopathy.

TGF-ß plays a pivotal role in the pathogenesis of GO by promoting orbital tissue remodeling and fibrosis. This process involves the stimulation of orbital fibroblasts, leading to myofibroblast differentiation, increased production of inflammatory mediators, and hyaluronan accumulation. Studies have elucidated TGF-ß's role in driving fibrosis and scarring processes through both canonical and non-canonical pathways, particularly resulting in the activation of orbital myofibroblasts and the excessive accumulation of extracellular matrix. Additionally, recent in vitro and in vivo studies have been summarized, highlighting the therapeutic potential of targeting TGF-ß signaling pathways, which may offer promising treatment interventions for GO. This review aims to consolidate the current understanding of the multifaceted role of TGF-ß in the molecular and cellular pathophysiology in Graves' ophthalmopathy (GO) by exploring its contributions to fibrosis, inflammation, and immune dysregulation. Additionally, the review investigates the therapeutic potential of inhibiting TGF-ß signaling pathways as a strategy for treating GO.

AMPK-mediated increase of glycolysis as an adaptive response to oxidative stress in human cells: implication of the cell survival in mitochondrial diseases.

We report that the energy metabolism shifts to anaerobic glycolysis as an adaptive response to oxidative stress in the primary cultures of skin fibroblasts from patients with MERRF syndrome. In order to unravel the molecular mechanism involved in the alteration of energy metabolism under oxidative stress, we treated normal human skin fibroblasts (CCD-966SK cells) with sub-lethal doses of H(2)O(2). The results showed that several glycolytic enzymes including hexokinase type II (HK II), lactate dehydrogenase (LDH) and glucose transporter 1 (GLUT1) were up-regulated in H(2)O(2)-treated normal skin fibroblasts. In addition, the glycolytic flux of skin fibroblasts was increased by H(2)O(2) in a dose-dependent manner through the activation of AMP-activated protein kinase (AMPK) and phosphorylation of its downstream target, phosphofructokinase 2 (PFK2). Moreover, we found that the AMPK-mediated increase of glycolytic flux by H(2)O(2) was accompanied by an increase of intracellular NADPH content. By treatment of the cells with glycolysis inhibitors, an AMPK inhibitor or genetic knockdown of AMPK, respectively, the H(2)O(2)-induced increase of NADPH was abrogated leading to the overproduction of intracellular ROS and cell death. Significantly, we showed that phosphorylation levels of AMPK and glycolysis were up-regulated to confer an advantage of survival for MERRF skin fibroblasts. Taken together, our findings suggest that the increased production of NADPH by AMPK-mediated increase of the glycolytic flux contributes to the adaptation of MERRF skin fibroblasts and H(2)O(2)-treated normal skin fibroblasts to oxidative stress.

The protective effect of antioxidants on orbital fibroblasts from patients with Graves' ophthalmopathy in response to oxidative stress.

PURPOSE: To investigate the biphasic effects of hydrogen peroxide (H2O2) on the orbital fibroblasts of patients with Graves' ophthalmopathy (GO) and the relation to antioxidants and proinflammatory cytokines. METHODS: Proliferation of cultured orbital fibroblasts from patients with GO and normal controls was evaluated in response to various concentrations of H2O2. The effect of low concentrations of H2O2 (6.25 µM) on the cellular proliferation and induction of intracellular proinflammatory cytokines, and reactive oxygen species of orbital fibroblasts were assessed. Protective effects of N-acetylcysteine and vitamin C on GO fibroblasts in response to 6.25 µM H2O2 stimulation were also investigated. RESULTS: When the GO fibroblasts were exposed to H2O2 at a concentration of 50 µM or above, significant cytotoxicity was observed. In contrast, lower concentrations of H2O2 (3.125-25 µM) increased the survival of GO fibroblasts with the peak cellular proliferation at 6.25 µM H2O2. However, this biphasic effect of H2O2 on the viability of orbital fibroblasts was not found in normal controls. In addition, 6.25 µM H2O2 led to significant elevation of the levels of transforming growth factor, beta 1, interleukin-1ß, and superoxide anion in GO fibroblasts, but no significant change in the normal controls. Pretreatment with N-acetylcysteine or vitamin C reversed the enhanced proliferation capacity and the induction of transforming growth factor, beta 1, interleukin-1ß and superoxide anion of GO fibroblasts in response to 6.25 µM H2O2. CONCLUSIONS: These findings revealed the biphasic effect of H2O2 on cellular proliferation of GO orbital fibroblasts. Importantly, a low level of H2O2 can stimulate proliferation of GO orbital fibroblasts and induce the production of proinflammatory cytokines, which can be inhibited by pretreatment with antioxidants. This provides a theoretical basis for the rational use of antioxidant in treating GO at an early stage.

Cadmium-based quantum dot induced autophagy formation for cell survival via oxidative stress.

Quantum dots (QDs) are one of most utilized nanomaterials in nanocrystalline semiconductors. QDs emit near-infrared fluorescence and can be applied as probes for detecting vasculature and imaging in biological systems. Since QDs have potential in clinical application, the toxicity of QDs needs to be carefully evaluated. In our present study, we elucidate the cytotoxic mechanisms of QDs using a mouse renal adenocarcinoma (RAG) cell line. QDs in RAG cells increased intracellular reactive oxygen species (ROS) levels and induced autophagy at 6 h, leading to subsequent apoptosis at 24 h. QDs entered the cells and were located within the endoplasmic reticulum (ER), endosome, and lysosome at 6 h and endosome, lysosome, and mitochondria at 24 h. However, QDs only affected mitochondrial function and did not induce ER stress. N-Acetylcysteine, an antioxidant agent, reduced intracellular ROS levels and decreased QD-induced autophagy but enhanced QD-induced cell death. Moreover, 3-methylamphetamine (an autophagy inhibitor) also reduced the cell viability in QD-treated cells. These findings suggest that ROS plays an essential role in the regulation of QD-induced autophagy, which subsequently enhances cell survival. Taken together, these results suggest that oxidative stress-induced autophagy is a defense/survival mechanism against the cytotoxicity of QD.

Molecular Mechanisms of UV-Induced Apoptosis and Its Effects on Skin Residential Cells: The Implication in UV-Based Phototherapy.

The human skin is an integral system that acts as a physical and immunological barrier to outside pathogens, toxicants, and harmful irradiations. Environmental ultraviolet rays (UV) from the sun might potentially play a more active role in regulating several important biological responses in the context of global warming. UV rays first encounter the uppermost epidermal keratinocytes causing apoptosis. The molecular mechanisms of UV-induced apoptosis of keratinocytes include direct DNA damage (intrinsic), clustering of death receptors on the cell surface (extrinsic), and generation of ROS. When apoptotic keratinocytes are processed by adjacent immature Langerhans cells (LCs), the inappropriately activated Langerhans cells could result in immunosuppression. Furthermore, UV can deplete LCs in the epidermis and impair their migratory capacity, leading to their accumulation in the dermis. Intriguingly, receptor activator of NF-κB (RANK) activation of LCs by UV can induce the pro-survival and anti-apoptotic signals due to the upregulation of Bcl-xL, leading to the generation of regulatory T cells. Meanwhile, a physiological dosage of UV can also enhance melanocyte survival and melanogenesis. Analogous to its effect in keratinocytes, a therapeutic dosage of UV can induce cell cycle arrest, activate antioxidant and DNA repair enzymes, and induce apoptosis through translocation of the Bcl-2 family proteins in melanocytes to ensure genomic integrity and survival of melanocytes. Furthermore, UV can elicit the synthesis of vitamin D, an important molecule in calcium homeostasis of various types of skin cells contributing to DNA repair and immunomodulation. Taken together, the above-mentioned effects of UV on apoptosis and its related biological effects such as proliferation inhibition, melanin synthesis, and immunomodulations on skin residential cells have provided an integrated biochemical and molecular biological basis for phototherapy that has been widely used in the treatment of many dermatological diseases.

Aberrant cell proliferation by enhanced mitochondrial biogenesis via mtTFA in arsenical skin cancers.

Arsenic-induced Bowen's disease (As-BD), a cutaneous carcinoma in situ, is thought to arise from gene mutation and uncontrolled proliferation. However, how mitochondria regulate the arsenic-induced cell proliferation remains unclear. The aim of this study was to clarify whether arsenic interfered with mitochondrial biogenesis and function, leading to aberrant cell proliferation in As-BD. Skin biopsy samples from patients with As-BD and controls were stained for cytochrome c oxidase (Complex IV), measured for mitochondrial DNA (mtDNA) copy number and the expression levels of mitochondrial biogenesis-related genes, including peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), nuclear respiratory factor 1 (NRF-1), and mitochondrial transcription factor A (mtTFA). The results showed that expression of cytochrome c oxidase, mtTFA, NRF-1, and PGC-1α was increased in As-BD compared with in healthy subjects. Treatment of primary keratinocytes with arsenic at concentrations lower than 1.0 µmol/L induced cell proliferation, along with enhanced mitochondrial biogenesis. Furthermore, we observed that the mitochondrial oxygen consumption rate and intracellular ATP level were increased in arsenic-treated keratinocytes. Blocking of mitochondrial function by oligomycin A (Complex V inhibitor) or knockdown of mtTFA by RNA interference abrogated arsenic-induced cell proliferation without affecting cyclin D1 expression. We concluded that mtTFA up-regulation, augmented mitochondrial biogenesis, and enhanced mitochondrial functions may contribute to arsenic-induced cell proliferation. Targeting mitochondrial biogenesis may help treat arsenical cancers at the stage of cell proliferation.

Reversing bladder outlet obstruction attenuates systemic and tissue oxidative stress.

UNLABELLED: What's known on the subject? and What does the study add? Oxidative damage in bladder tissue and systemic oxidative biomarkers were both found to be increased in rabbits with partial bladder outlet obstruction. It is shown that the reversal of partial bladder outlet obstruction will attenuate the systemic oxidative stress. OBJECTIVE: To investigate whether partial bladder outlet obstruction (PBOO) increases systemic oxidative stress and whether relief of PBOO could attenuate this stress. MATERIALS AND METHODS: Surgically created PBOO in male New Zealand white rabbits was assessed after 4 weeks in one group of rabbits (n = 4), and was relieved in two additional groups of rabbits (n = 4 each) that were assessed at 4 and 8 weeks after relief of PBOO. Four sham-operated rabbits served as controls. The assessed oxidative stress biomarkers included urinary and plasma 8-hydroxy-2'-deoxyguanosine (8-OHdG) and plasma malondialdehyde (MDA), total anti-oxidant capacity (TAC) and glutathione (GSH). In addition, the copy number of mitochondrial DNA and the 8-OHdG content in bladder tissues from these rabbits were also determined at the beginning and at indicated time points in the experiments. RESULTS: There were significant increases in both the 8-OHdG levels of urine, plasma and bladder tissue and the plasma MDA after induction of PBOO. There were also significant decreases in the TAC, in GSH levels and in mitochondrial DNA copy number in bladder tissues after PBOO. Most importantly, all of the values returned toward the control levels after the PBOO was reversed at 8 weeks. CONCLUSION: PBOO increases systemic and oxidative stress and its reversal results in a progressive reduction of both systemic and tissue oxidative stress.

Increased response to oxidative stress challenge in Graves' ophthalmopathy orbital fibroblasts.

PURPOSE: To investigate whether orbital fibroblasts from patients with Graves' ophthalmopathy (GO) are more responsive to oxidative stress. METHODS: Lipid peroxidation, oxidative DNA damage, reactive oxygen species (ROS) contents and activities of antioxidant enzymes were measured in cultured orbital fibroblasts from GO patients and age-matched normal controls in response to 200 µM hydrogen peroxide (H(2)O(2)). RESULTS: GO fibroblasts had increased basal levels of malondialdehyde (MDA), 8-hydroxy 2'-deoxyguanosine, superoxide anions, H(2)O(2), and manganese-dependent superoxide dismutase (Mn-SOD) activity, as well as decreased glutathione peroxidase (GPx) activity and the ratio between reduced (GSH) and oxidized glutathione (GSSG) compared with the orbital fibroblasts from normal subjects. After treatment of the cells with 200 µM H(2)O(2), the amplitude of increase in the intracellular levels of MDA (63% versus 26%), H(2)O(2) (24% versus 13%) and Mn-SOD activity (48% versus 23%) was exaggerated in GO fibroblasts compared with normal controls, respectively. In addition, treatment of GO fibroblasts with 200 µM H(2)O(2) led to a dramatic reduction of catalase activity (-59% versus -29%), GPx activity (-56% versus -13%), and GSH/GSSG ratio (-49% versus -21%), respectively. CONCLUSIONS: Elevated ROS and redox imbalance in GO orbital fibroblasts were exacerbated by H(2)O(2) as a result of exhaustion of GSH and compromise of antioxidant enzymes. Hypersensitivity to oxidative stress of GO orbital fibroblasts may play a role in the pathogenesis of GO.

Oxidative stress biomarkers in urine and plasma of rabbits with partial bladder outlet obstruction.

UNLABELLED: What's known on the subject? and What does the study add? It has been known that there is an increase of oxidative damage in the bladder tissues of animals after PBOO. However, no reliable oxidative stress biomarkers in either urine or plasma have been available for the assessment of the severity of PBOO. This study clearly demonstrated that the levels of oxidative stress biomarkers are increased in urine and plasma of the rabbits with PBOO. OBJECTIVE: To investigate oxidative stress and oxidative damage biomarkers in urine and plasma after partial bladder outlet obstruction (PBOO) in rabbits. MATERIALS AND METHODS: In all, 16 male New Zealand White rabbits were separated equally into four groups: a control group and PBOO-treated groups for 2, 4 and 8 weeks. The oxidative stress biomarkers assessed included urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) and plasma malondialdehyde (MDA). We also measured the total antioxidant capacity (TAC) in blood plasma. 8-OHdG, MDA and TAC were measured at both the beginning and indicated time points of the experimental design. RESULTS: There was no significant difference in body weight among rabbits in the four groups. However, there was a significant increase in bladder weight after 2 weeks of PBOO. After 4 and 8 weeks of PBOO, there was an additional significant increase in bladder weight in all three groups. There was no difference in blood creatinine levels among the groups. In the 4- and 8-week PBOO groups, there was a significant increase of 8-OHdG in urine and of MDA in plasma, while there was a significant decrease in TAC in plasma. CONCLUSION: The results showed that oxidative stress could be detected in the plasma and urine of rabbits after 4 and 8 weeks of PBOO, and not only from bladder tissue as previously reported. Thus, there could be an easy and alternative way to evaluate bladder function by analysis of urine and/or plasma. Additionally, rabbits with chronic PBOO showed an increase in systemic oxidative stress, which could be a novel starting point for examining the link between the lower urinary tract symptoms/benign prostate hyperplasia and metabolic syndrome in future studies.

Effect of Pirfenidone on TGF-ß1-Induced Myofibroblast Differentiation and Extracellular Matrix Homeostasis of Human Orbital Fibroblasts in Graves' Ophthalmopathy.

Pirfenidone is a pyridinone derivative that has been shown to inhibit fibrosis in animal models and in patients with idiopathic pulmonary fibrosis. Its effect on orbital fibroblasts remains poorly understood. We investigated the in vitro effect of pirfenidone in transforming growth factor-ß1 (TGF-ß1)-induced myofibroblast transdifferentiation and extracellular matrix (ECM) homeostasis in primary cultured orbital fibroblasts from patients with Graves' ophthalmopathy (GO). The expression of fibrotic proteins, including α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), fibronectin, and collagen type I, was determined by Western blots. The activities of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) responsible for the ECM homeostasis were examined. After pretreating the GO orbital fibroblasts with pirfenidone (250, 500, and 750 µg/mL, respectively) for one hour followed by TGF-ß1 for another 24 h, the expression of α-SMA, CTGF, fibronectin, and collagen type I decreased in a dose-dependent manner. Pretreating the GO orbital fibroblasts with pirfenidone not only abolished TGF-ß1-induced TIMP-1 expression but recovered the MMP-2/-9 activities. Notably, pirfenidone inhibited TGF-ß1-induced phosphorylation of p38 and c-Jun N-terminal kinase (JNK), the critical mediators in the TGF-ß1 pathways. These findings suggest that pirfenidone modulates TGF-ß1-mediated myofibroblast differentiation and ECM homeostasis by attenuating downstream signaling of TGF-ß1.

The Role of Oxidative Stress and Therapeutic Potential of Antioxidants in Graves' Ophthalmopathy.

Graves' ophthalmopathy (GO) is the most common extrathyroidal manifestation of Graves' disease. It is characterized initially by an inflammatory process, followed by tissue remodeling and fibrosis, leading to proptosis, exposure keratopathy, ocular motility limitation, and compressive optic neuropathy. The pathogenic mechanism is complex and multifactorial. Accumulating evidence suggests the involvement of oxidative stress in the pathogenesis of GO. Cigarette smoking, a major risk factor for GO, has been shown to induce reactive oxygen species (ROS) generation and oxidative damage in GO orbital fibroblasts. In addition, an elevation in ROS and antioxidant enzymes is observed in tears, blood, and urine, as well as orbital fibroadipose tissues and fibroblasts from GO patients. In vitro and in vivo studies have examined the efficacy of various antioxidant supplements for GO. These findings suggest a therapeutic role of antioxidants in GO patients. This review summarizes the current understanding of oxidative stress in the pathogenesis and potential antioxidants for the treatment of GO.

Response to the increase of oxidative stress and mutation of mitochondrial DNA in aging.

In the aging process, mitochondrial function gradually declines with an increase of mutations in mitochondrial DNA (mtDNA) in tissue cells. Some of the aging-associated mtDNA mutations have been shown to result in not only inefficient generation of ATP but also increased production of reactive oxygen species (ROS) such as superoxide anions (O2-) and hydrogen peroxide (H2O2) in the mitochondria of aging tissues. Extensive studies have revealed that such an increase of oxidative stress is a contributory factor for alterations in the expression and activities of antioxidant enzymes and increased oxidative damage to DNA, RNA, proteins, and lipids in tissues and cultured cells from elderly subjects. Recently, we observed that gene expression of several proteins and enzymes related to iron metabolism is altered and that aconitase is extremely susceptible to oxidative damage in senescent skin fibroblasts and in cybrids harboring aging-associated A8344G mutation of mtDNA. Of great importance is the perturbation at the protein and activity levels of several enzymes containing iron-sulfur clusters in skin fibroblasts of elderly subjects. Taken together, these findings suggest that cellular response to oxidative stress and oxidative damage elicited by mitochondrial dysfunction and/or mtDNA mutations plays an important role in human aging.

The difference in oxidative stress of the blood between using 5% glucose water and distilled water as the irrigant for BPH patients undergoing transurethral resection of the prostate.

INTRODUCTION: To evaluate the difference in oxidative stress in the blood between using 5% glucose water and using distilled water as the irrigant for BPH patients receiving transurethral resection of the prostate (TURP), we conducted this prospective study, on a total of 38 patients with symptomatic BPH. MATERIALS AND METHODS: All the patients were randomly divided into 2 groups. Distilled water in group A and 5% glucose water in group B were used as the irrigant. The oxidative stress and antioxidant capacity were evaluated by measuring malondialdehyde (MDA), mitochondrial DNA (mtDNA) copy number and total capacity of antioxidants (TOA) in the blood. The data were correlated with serum creatinine and sodium, which were measured before and immediately after TURP in all patients. RESULTS: Serum creatinine increased from 0.86 +/- 0.17 to 1.02 +/- 0.20 mg/dL while serum sodium decreased from 139.7 +/- 2.3 to 136.4 +/- 4.0 mEq/L immediately after surgery in patients of group A (p < 0.05). MDA was increased and TOA decreased in all the patients immediately after TURP. Patients in group A had significantly higher MDA, lower mtDNA copy number, higher degree of oxidative mtDNA damage and lower TOA than those in group B immediately after TURP. CONCLUSION: In conclusion, we observed an increase of MDA and decrease of TOA in blood in all the patients immediately after TURP, which might induce renal damage and decrease serum sodium. Moreover, the oxidative stress levels in blood of patients in group A were significantly higher than those in group B immediately after TURP.

Essential role of connective tissue growth factor (CTGF) in transforming growth factor-ß1 (TGF-ß1)-induced myofibroblast transdifferentiation from Graves' orbital fibroblasts.

Connective tissue growth factor (CTGF) associated with transforming growth factor-ß (TGF-ß) play a pivotal role in the pathophysiology of many fibrotic disorders. However, it is not clear whether this interaction also takes place in GO. In this study, we investigated the role of CTGF in TGF-ß-induced extracellular matrix production and myofibroblast transdifferentiation in Graves' orbital fibroblasts. By Western blot analysis, we demonstrated that TGF-ß1 induced the expression of CTGF, fibronectin, and alpha-smooth muscle actin (α-SMA) in Graves' orbital fibroblasts. In addition, the protein levels of fibronectin and α-SMA in Graves' orbital fibroblasts were also increased after treatment with a recombinant human protein CTGF (rhCTGF). Moreover, we transfected the orbital fibroblasts with a small hairpin RNA of CTGF gene (shCTGF) to knockdown the expression levels of CTGF, which showed that knockdown of CTGF significantly diminished TGF-ß1-induced expression of CTGF, fibronectin and α-SMA proteins in Graves' orbital fibroblasts. Furthermore, the addition of rhCTGF to the shCTGF-transfected orbital fibroblasts could restore TGF-ß1-induced expression of fibronectin and α-SMA proteins. Our findings demonstrate that CTGF is an essential downstream mediator for TGF-ß1-induced extracellular matrix production and myofibroblast transdifferentiation in Graves' orbital fibroblasts and thus may provide with a potential therapeutic target for treatment of GO.

Expression and clinical significance of connective tissue growth factor (CTGF) in Graves' ophthalmopathy.

AIMS: To examine the expression of connective tissue growth factor (CTGF) in human cultured orbital fibroblasts from patients with Graves' ophthalmopathy (GO) and investigate whether a correlation exists between the presence of CTGF protein and clinical parameters of the disease. METHODS: The protein expression levels of CTGF were analysed by western blots in cultured orbital fibroblasts from 10 patients with GO and 7 age-matched normal controls. Associations between the protein expression of CTGF and the clinical factors of GO, including clinical demographics, thyroid function, clinical activity score (CAS) and ophthalmopathy index (OI), was evaluated. RESULTS: The mean protein expression levels of CTGF in the GO orbital fibroblasts were significantly higher than those of normal controls (p<0.001). Based on further analysis, the protein expression levels of CTGF in the GO orbital fibroblasts had significant correlation with gender (p=0.029), serum levels of thyrotropin receptor antibodies (p=0.029), CAS (p=0.048) and OI (p=0.043). Especially, there was a significant correlation between protein expression levels of CTGF and lid oedema (p=0.037), proptosis (p=0.045) and corneal involvement (p=0.001). CONCLUSIONS: Our findings revealed that the protein expression levels of CTGF in the GO orbital fibroblasts were significantly highly expressed than those of normal controls, and the elevated CTGF was associated with clinical characteristics and evolution, indicating CTGF may play a role in the pathogenesis and pathophysiology of GO.